Medical devices incorporating functional adhesives

ABSTRACT

A method for bonding a polymeric medical device to tissue is provided which includes providing a polymeric medical device having a plurality of reactive members of a specific binding pair attached on a surface of the medical device, and providing tissue with a plurality of complementary reactive members of the specific binding pair, wherein upon contact of the reactive members on the surface of the medical device with the complimentary reactive members on the tissue, covalent bonds are formed between the reactive members and the complementary reactive members, thus adhering the device to the tissue. A kit is provided including a polymeric medical device such as a patch or mesh having a plurality of reactive members of a specific binding pair attached to a surface of the device and an applicator containing a solution or suspension of complementary reactive members of the specific binding pair, the complementary reactive members having a functionality that will adhere them to biological tissue upon contact, said applicator adapted to deliver the solution or suspension to biological tissue.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/154,367 filed Feb. 21, 2009.

BACKGROUND

1. Technical Field

The present disclosure relates to adhesive modalities for repair ofbiological tissues.

2. Related Art

Techniques for repairing damaged or diseased tissue are widespread inmedicine. Wound closure devices such as sutures, staples and otherrepair devices such as mesh or patch reinforcements are frequently usedfor repair. Surgical adhesives have been used to augment and, in somecases, replace sutures and staples in wound closure. For example, in thecase of hernias, techniques involving the use of a mesh or patch toreinforce the abdominal wall are being used. The mesh or patch cangenerally be held in place by suturing or stapling to the surroundingtissue. Unfortunately, the use of such sutures or staples may increasethe patient's discomfort and, in certain instances, there may be a riskof weakening thin or delicate tissue where they are attached. Certaintechniques involve placing a mesh or patch against the repair sitewithout suturing or stapling, e.g., allowing the pressure of theperitoneum to hold the patch against the posterior side of the abdominalwall. However, fixation of the mesh or patch is generally preferred inorder to avoid folding, shrinkage, and migration of the mesh or patch.Surgical adhesives such as cyanoacrylates and fibrin glues have beenused as fixatives in lieu of, or in addition to, suturing or staplingthe mesh or patch. However, fibrin adhesives can be difficult to prepareand store. Cyanoacrylates may cause irritation at the point ofapplication and may not provide a sufficient degree of elasticity. Inaddition, surgical adhesives can tend to form a physical barrier betweenthe item or items being attached to biological tissue, thus interferingwith tissue ingrowth into the item when ingrowth is desired.

Click chemistry is a popular term for reliable reactions that make itpossible for certain chemical building blocks to “click” together andform an irreversible linkage. See, e.g., US Pub. No. 2005/0222427. Sinceits recent introduction, click chemistry has been used for ligation inbiological and medical technology. In the case of azide-alkyne clickchemistry, the reactions may be catalyzed or uncatalyzed. For example,copper-free click chemistry was recently developed by Bertozzi andcolleagues using difluorinated cyclooctyne or DIFO, that reacts withazides rapidly at physiological temperatures without the need for atoxic catalyst. See, e.g., Baskin et al., Copper Free Click Chemistryfor Dynamic In Vivo Imaging, PNAS, vol. 104, no. 43, 16793-16797 (Oct.23, 2007). The critical reagent, a substituted cyclooctyne, possessesring strain and electron-withdrawing fluorine substituents that togetherpromote a [3+2] dipolar cycloaddition with azides. See also, US Pub. No.2006/0110782 and Codelli et al., Second Generation DifluorinatedCyclooctynes for Copper-Free Click Chemistry, J. Am. Chem. Soc., vol.130, no. 34, 11486-11493 (2008). Another suitable cyclooctyne is6,7-dimethoxyazacyclooct-4-yne (DIMAC). See, Sletton and Bertozzi, Ahydrophilic azacyclooctyne for Cu-free click chemistry, Org. Lett.(2008) 10 (14), 3097-3099. Other click chemistry reactions includeDiels-Alder reactions, thiol-alkene reactions, and maleimide-thiolreactions. There is a continuing need to generate improvements in tissuerepair technology and advance the state of the art.

SUMMARY

A method for bonding a polymeric medical device to biological tissue isprovided which includes providing a polymeric medical device having aplurality of reactive members of a specific binding pair attached on asurface of the medical device, and providing tissue with a plurality ofcomplementary reactive members of the specific binding pair, whereinupon contact of the reactive members on the surface of the medicaldevice with the complimentary reactive members on the tissue, covalentbonds are formed between the reactive members and the complementaryreactive members, thus bonding the device to the tissue.

DETAILED DESCRIPTION

A surgical bonding system is provided which covalently bonds reactivemembers of a specific binding pair to one another via click chemistry.Click chemistry refers to a collection of reactive members having a highchemical potential energy capable of producing highly selective, highyield reactions. The reactive members react to form extremely reliablemolecular connections in most solvents, including physiologic fluids,and often do not interfere with other reagents and reactions. Examplesof click chemistry reactions include Huisgen cycloaddition, Diels-Alderreactions, thiol-alkene reactions, and maleimide-thiol reactions.

Huisgen cycloaddition is the reaction of a dipolarophile with a1,3-dipolar compound that leads to 5-membered (hetero)cycles. Examplesof dipolarophiles are alkenes and alkynes and molecules that possessrelated heteroatom functional groups (such as carbonyls and nitriles).1,3-Dipolar compounds contain one or more heteroatoms and can bedescribed as having at least one mesomeric structure that represents acharged dipole. They include nitril oxides, azides, and diazoalkanes.Metal catalyzed click chemistry is an extremely efficient variant of theHuisgen 1,3-dipolar cycloaddition reaction between alkyl-aryl)-sulfonylazides, C—N triple bonds and C—C triple bonds which is well-suitedherein. The results of these reactions are 1,2 oxazoles, 1,2,3 triazolesor tetrazoles. For example, 1,2,3 triazoles are formed by a coppercatalyzed Huisgen reaction between alkynes and alkyl/aryl azides. Metalcatalyzed Huisgen reactions proceed at ambient temperature, are notsensitive to solvents, i.e., nonpolar, polar, semipolar, and are highlytolerant of functional groups. Non-metal Huisgen reactions (alsoreferred to as strain promoted cycloaddition) involving use of asubstituted cyclooctyne, which possesses ring strain andelectron-withdrawing substituents such as fluorine, that togetherpromote a [3+2] dipolar cycloaddition with azides are especiallywell-suited for use herein due to low toxicity as compared to the metalcatalyzed reactions. Examples include DIFO and DIMAC. Reaction of thealkynes and azides is very specific and essentially inert against thechemical environment of biological tissues. One reaction scheme may berepresented as:

where R and R′ are a polymeric material or a component of a biologictissue.

The Diels-Alder reaction combines a diene (a molecule with twoalternating double bonds) and a dienophile (an alkene) to make rings andbicyclic compounds.

Examples include:

The thiol-alkene (thiol-ene) reaction is a hydrothiolation, i.e.,addition of RS—H across a C═C bond. The thiol-ene reaction proceeds viaa free-radical chain mechanism. Initiation occurs by radical formationupon UV excitation of a photoinitiator or the thiol itself. Thiol-enesystems form ground state charge transfer complexes and thereforephotopolymerize even in the absence of initiators in reasonablepolymerization times. However, the addition of UV light increases thespeed at which the reaction proceeds. The wavelength of the light can bemodulated as needed, depending upon the size and nature of theconstituents attached to the thiol or alkene. A general thiol-enecoupling reaction mechanism is represented below:

In accordance with the disclosure herein, a polymeric medical device,such as a surgical patch or mesh is provided with a plurality ofreactive members of a specific binding pair attached on the surface ofthe medical device. When the reactive members of the medical device arecontacted with biological tissue containing complementary reactivemembers of the specific binding pair, covalent attachment occurs, thusadhering the device to the tissue. In embodiments, the reactive membersmay be either a dipolarophile or a 1,3 dipolar compound depending onwhich complement is applied to the target tissue or the medical device.For example, if a dipolarphile is located on the device, the 1,3 dipolarcompound will be located on the tissue. If a dipolarphile is located onthe tissue, the 1,3 dipolar compound will be located on the device. Inembodiments, the Diels-Alder members of a specific binding pair may beeither a diene and a dienophile depending on which complement is appliedto the target tissue or the medical device. For example, if a diene islocated on the device, the dienophile can be located on the tissue. If adiene is located on the tissue, the dienophile can be located on thedevice. In embodiments, the thiol-ene members of a specific binding pairmay be either a thiol and an alkene depending on which complement isapplied to the target tissue or the device. For example, if a thiol islocated on the device, the alkene can be located on the tissue. If athiol is located on the tissue, the alkene can be located on the device.

The polymeric device may be constructed from biocompatible absorbablepolymers or biocompatible non-absorbable polymers. Examples of suitablepolymers include polycarbonates, polyolefins, polymethacrylates,polystyrenes, polyamides, polyurethanes, polyethylene terephthalate,poly (lactic acid), poly (glycolic acid), poly (hydroxbutyrate),dioxanones (e.g., 1,4-dioxanone), δ-valerolactone, 1,dioxepanones (e.g.,1,4-dioxepan-2-one and 1,5-dioxepan-2-one), poly (phosphazine),polyesters, polyethylene glycol, polyethylene oxides, polyacrylamides,cellulose esters, fluoropolymers, vinyl polymers, silk, collagen,alginate, chitin, chitosan, hyaluronic acid, chondroitin sulfate,glycosaminoglycans, polyhydroxyethylmethylacrylate,polyvinylpyrrolidone, polyvinyl alcohol, polyacrylic acid, polyacetate,polycaprolactone, polypropylene, glycerols, poly(amino acids), copoly(ether-esters), polyalkylene oxalates, polyamides, poly(iminocarbonates), polyalkylene oxalates, polyoxaesters,polyorthoesters, polyphosphazenes, polypeptides and copolymers, blockcopolymers, homoploymers, blends and combinations thereof.

In the present application, the term “bioresorbable” and “bioabsorbable”are used interchangeably and are intended to mean the characteristicaccording to which an implant and/or a material is resorbed by thebiological tissues and the surrounding fluids and disappears in vivoafter a given period of time, that may vary, for example, from one dayto several months, depending on the chemical nature of the implantand/or of the material. Non bioresorbable material—also called permanentmaterial—is not substantially resorbed by tissues and surroundingfluids, after 2 years and more, keeping in particular most (e.g., >80%)of their mechanical properties after such a time. The term“biocompatible” is intended to mean the characteristic according towhich an implant and/or a material is well integrated by the biologicaltissues and the surrounding fluids without inducing excessiveinflammation reaction around the bulk of the material or due to itsdegradation. The material should avoid also the formation of a fibrouscapsule which usually results in the delay of the cellular integrationof a porous implant.

Many of the above described examples of polymers do not containfunctional groups in their molecules. In embodiments, the reactivemembers are attached to the medical device by surface modificationtechniques such as plasma treatment, silane coupling treatment and acidsensitization. Surface activation of the medical device can be achievedby acid or base hydrolysis, treatment by means of cold plasma, bychemical reactions or electromagnetic radiations.

Hydrolysis can be conducted in the presence of an aqueous solution of abase or an acid to accelerate surface reaction, inasmuch as excessivelylong processes of activation can induce a reduction in molecular weightand thus in the mechanical properties of the material. Suitable basesfor obtaining watery solutions suited to the aim are, for example,strong alkalis, such as LiOH, Ba(OH)₂, Mg(OH)₂, NaOH, KOH, Na₂CO₃,Ca(OH)₂ and the weak bases, such as for example NH₄ OH and the aminessuch as methylamine, ethylamine, diethylamine and dimethylamine. Acidssuitable for surface hydrolysis treatments can be chosen, for example,from among HCl, HClO₃, HClO₄, H₂SO₃, H₂ SO₄, H₃ PO₃, H₃ PO₄, HI, HIO₃,HBr, lactic acid, glycolic acid. Surface activation by means ofhydrolysis can be conducted at temperatures preferably comprised between0 degrees Celsius and the material softening temperature.

Plasma treatment can be carried out both in the presence of a reactivegas, for example air, Ar, O₂ with the formation of surface activation ofoxygenate type, such as —OH, —CHO, —COOH.

Surface treatment, whether hydrolytic or with plasma, can remainunaltered or can be followed by further chemical modifications toprovide the first reactive groups on the bioabsorbable polymericsubstrate. Thus, for example, the COONa groups generated by a basehydrolysis can be subsequently converted into COOH groups by treatmentwith strong mineral acids. Further, the surface freeing of alcoholicgroups by means of a hydrolysis process can be followed by reaction bymeans of the addition of a compound provided with functional group orgroups able to react with surface alcoholic groups, such as for exampleby means of the addition of an anhydride such as succinic anhydride,with the conversion of —OH groups into —O—CO—CH₂—CH₂—COOH groups.Suitable surface activation techniques are disclosed in U.S. Pat. No.6,107,453, the entire disclosure of which is incorporated herein by thisreference.

During manufacture of polymers, pendant functional groups can beincorporated into the polymer backbone by, e.g., copolymerization withfunctionalized monomer such as lactones, cyclic carbonates andmorpholine-2,5-diones. The azido group, N₃ is a nucleophilic group thatwill exchange with other nucleophilic groups, e.g., —OH, —NH₂ andhalogens (Br, Cl, or I). For example, 1,3-dipolar compounds may beconjugated to aliphatic polyesters, by copolymerizing ε-caprolactone andα-chloro-ε-caprolactone and then substituting an azide group for the Clatom. Polyesters can incorporate pendant dipolarophiles, e.g., propargylgroups, by copolymerization of ε-caprolactone andα-propargyl-δ-valerolactone. Copolymers of L-lactide containingpropargyl groups may, e.g., be prepared by ring opening copolymerizationof 5-methyl-5-propargyloxycarbonyl-1,3-dioxanone with L-lactide at amolar ratio of about 90:10 with ZnEt₂ as a catalyst. See, Shi et al.,Biomaterials, 29 (2008)1118-1126. Azide functionalized polystyrene issynthesized using atom transfer radical polymerization and subsequentmodification with azidotrimethylsilane and tetrabutylammonium fluoride.See, Dirks, et al., Chem. Comm., (2005) 4172-4174. Azides may beincorporated onto methacrylates, e.g., 3 azidopropyl methacrylate whichis copolymerized to a block copolymer. Diels-Alder functionalities andthiol-ene functionalities are likewise incorporated into polymersherein.

In embodiments, the medical device is a surgical patch. The surgicalpatch may be selected from any conventional patch type that is suitablefor use in tissue reinforcement, e.g., hernia repair, or as ananti-adhesion barrier, hemostatic patch, bandages, pledgets and thelike. Many types of patches are currently available and are well knownto those skilled in the art. Exemplary polymeric patch materials includenonabsorbable polyester cloth, polyester sheeting, acrylic cloth,polyvinyl sponge or foam, polytetrafluoroethylene (PTFE), expanded PTFE,and polyvinyl cloth. Any of the biocompatible polymers listed above maybe utilized. In another embodiment, the medical device is a surgicalmesh, e.g., polypropylene mesh, nylon mesh, and Dacron mesh. Exemplaryabsorbable meshes include collagen, polyglycolic acid, polyglactin,polycaprolactone, chitosan, and carbon fiber mesh. It should beunderstood that any of the above-mentioned biocompatible polymers may besuitable for use herein.

Indeed, the patch or mesh may be produced from fibers of anybiocompatible polymer using any techniques known to those skilled in theart, such as knitting, weaving, tatting, nonwoven techniques, freezedrying, solvent casting and the like. It is envisioned that the patch ormesh may be formed from any permanent biocompatible materials (e.g.polyesters, polypropylene), biodegradable biocompatible materials (e.g.polylactic acid, polyglycolic acid, oxidized cellulose, and chitosan) orwith a combination at any proportion of both permanent and biodegradablematerials. The medical device may, for example, have an openworkthree-dimensional (“3D”) structure (see, e.g. U.S. Pat. No. 6,451,032,the entire disclosure of which is incorporated herein by reference),e.g., a “honeycomb” structure, and thus a certain thickness whichseparates the two surfaces of the fabric.

In certain embodiments, the patch is composed of a biopolymer foamhaving openings or pores over at least a portion of a surface thereof.The pores may be in sufficient number and size so as to interconnectacross the entire thickness of the porous layer. Alternatively, thepores may not interconnect across the entire thickness of the porouslayer. Closed cell foams are illustrative examples of structures inwhich the pores may not interconnect across the entire thickness of theporous layer. In yet other embodiments, the pores do not extend acrossthe entire thickness of the foam, but rather are present at a portion ofthe surface thereof. In some embodiments, the openings or pores arelocated on a portion of the surface of the porous layer, with otherportions of the porous layer having a non-porous texture. Those skilledin the art may envision other pore distribution patterns andconfigurations for the foam.

In certain embodiments, the foam may be made from non-denatured collagenor collagen which has at least partially lost its helical structurethrough heating or any other known method, consisting mainly ofnon-hydrolyzed a chains, and having a molecular weight, in embodiments,of about 100 kDa. The collagen used for the porous layer of the presentdisclosure may be native collagen or atelocollagen, which may beobtained via pepsin digestion and/or after moderate heating as definedhereinabove. The origin and type of collagen may be as indicated for thenon-porous layer described hereinabove.

The collagen may be cured to any desired degree. The collagen suspensionor solution may be made from non-cured, moderately cured, highly curedor extremely highly cured collagens or combinations thereof at anyproportions. As used herein, the term “moderately cured” is intended tomean that the degradation of the porous layer will be at least about 90%complete (as measured by residual weight) by the end of about threeweeks of implantation; the term “highly cured” is intended to mean thatthe degradation of the porous layer will be at least about 90% complete(as measured by residual weight) by the end of about three months ofimplantation; and the term “extremely highly cured” is intended to meanthat the degradation of the porous layer will be at least about 90%complete (as measured by residual weight) by the end of about two yearsof implantation.

Moderately cured collagen may be obtained by oxidative cleavage ofcollagen by periodic acid or one of its salts, as described forcollagens of the non-porous layer. In embodiments, highly cured collagenmay be made from collagen cross-linked by glutaratdehyde or by any otherknown cross-linking agents such as, for example, but not limited to,isocyanates. The degree of crosslinking distinguishes between highlycured and very highly cured materials. Techniques for curing to variousdegrees are within the purview of those skilled in the art.

In certain embodiments, the collagen may optionally include noncollagenic components, such as glycosaminoglycans, for example, but notlimited to, chitosan. The glycosaminoglycans, in embodiments, display adegree of acetylation (DA) of from about 0.5% to about 50%, have amolecular weight ranging from about 100 g/mol to about 1,000,000 g/mol,and may display a low polydispersity index of from about 1 to about 2.In certain embodiments, the collagen may be a mixture of chitosans andother glycosamoniglycans, for example, but not limited to, hyaluronicacid, which, after deacettylation have free amino groups capable ofcross-linking to the oxidized collagen. It is contemplated that thecollagen suspension or solution may be a combination of oxidizedcollagen and chitosan which can form a cross-linked network. In certainembodiments, patch or mesh may be formed from one or more bioresorbable,natural biological polymers. Suitable natural biological polymersinclude, but are not limited to, collagen, gelatin, cellulose,hydroxypropyl cellulose, carboxyethyl cellulose, chitin, chitosan,hyaluronic acid, chondroitin sulfate and other gycosaminoglycans andcombinations thereof. In alternate embodiments, the polymer constituentmay be a polysaccharide such as chitin or chitosan, or polysaccharidesmodified by oxidation of alcohol functions into carboxylic functionssuch as oxidized cellulose. It is contemplated that the naturalbiological polymers may be combined with any biocompatible syntheticmaterials to produce the porous layer of the implant.

Biological tissue is provided with reactive members of a specificbinding pair by conjugation to various components of tissue such asproteins, lipids, oligosaccharides, oligonucleotides, glycans, includingglycosaminoglycans. In one embodiment, the reactive members orcomplementary reactive members are attached directly to components ofthe tissue. In another embodiment, the reactive members or complementaryreactive members are attached to components of the tissue via a linker.In either case, situating the reactive members or complementary reactivemembers on the tissue can be accomplished by suspending the reactivemembers or complementary reactive members in a solution or suspensionand applying the solution or suspension to the tissue such that thereactive member binds to a target. The solution or suspension may bepoured, sprayed or painted onto the tissue, whereupon the reactivemembers or complementary reactive members are incorporated into thetissue.

1,3-Dipolar compounds can be incorporated into proteins, lipids,oligosaccharides, oligonucleotides and glycans using, e.g., metabolicmachinery, covalent inhibitors and enzymatic transfers. For example, anazido group, N₃, can be applied at the N-terminus of proteins orpeptides using azidoacetyl chloride. See, e.g., Haridas, et al.,Tetrahedron Letters 48 (2007) 4719-4722. The azido group is anucleophilic group that will exchange with other nucleophilic groups,e.g., —OH, —NH₂ and halogens (Br, Cl, or I). NaN₃ is an azidizing agentwhich is capable of aziding proteins by simply contacting the proteinswith a 10 times molar excess of NaN₃. A process for C-terminalazidization is described in Cazalis, et al., Bioconjugate Chem., 15(2004) 1005-1009. Incubation of cells with peracetylatedN-azidoacetylmannosamine provides cell surface glycans with azido sialicacid. See, e.g., Codelli et al., J. Amer. Chem. Soc., 130 (34)11486-11493 (2008). Azido-tagged lipids are described in Smith, et al.,Bioconjugate Chem., 19 (9), 1855-1863 (2008). PEGylation is a commonlyused technique for adding groups to to peptides and proteins and issuitable for use herein. For example, PEG may be covalently bound toamino acid residues via a reactive group. Reactive groups (as opposed toreactive members herein) are those to which an activated PEG moleculemay be bound (e.g., a free amino or carboxyl group). For example,N-terminal amino acid residues and lysine (K) residues have a free aminogroup and C-terminal amino acid residues have a free carboxyl group.Sulfhydryl groups (e.g., as found on cysteine residues) may also be usedas a reactive group for attaching PEG. In addition, enzyme-assistedmethods for introducing activated groups (e.g., hydrazide, aldehyde, andaromatic-amino groups) specifically at the C-terminus of a polypeptide.Accordingly, PEG incorporating 1,3-dipolar compounds may be utilizedherein. Those skilled in the art can utilize any known process forcoupling a 1,3-dipolar compound into proteins, lipids, oligosaccharides,oligonucleotides and glycans.

Dipolarophile functionalized proteins and peptides can be synthesized bylinking at the N-terminus with, for example, an alkyne (e.g., 3 butynylchloroformate), in connection with a tripeptide (GlyGlyArg). See, Dirks,et al., supra. A suitable tripeptide herein is the well-known celladhesion sequence RGD. It should be understood that, as used herein,“proteins” is intended to encompass peptides and polypeptides. In oneembodiment, thiols on cysteines are functionalized with alkyne bearingmaleimide. Id. Providing a C-terminal dipolarophile can be accomplished,e.g., by coupling with propargylamine using a cross-linking agent suchas N-hydroxysuccinimide/DCC. See, e.g., Haridas, et al. supra. Terminalalkynes can be installed using metabolic building blocks such asalkynoic acids. Lipids may be functionalized with alkynes. For example,alkyne modified fatty acids can be generated by reaction of terminalalkynyl-alkyl bromide with trimethyl phosphine to yield a 16 carbonalkynyl-dimethylphosphonate. See, e.g., Raghavan et al., Bioorg. Med.Chem. Lett., 18 (2008) 5982-5986. As above, PEGylation may be used foradding dipolarophile groups to peptides and proteins and is suitable foruse herein. Diels-Alder functionalities and thiol-ene functionalitiesare likewise attached to proteins, lipids, oligosaccharides,oligonucleotides and glycans.

The reactive members or complementary reactive members may be alsoattached to biological tissue via a linker. In certain embodiments, thelinker is or includes a ligand which bears a reactive member. The ligandbinds to a desired target on the tissue and thus provides a vehicle fortransporting and indirectly binding the reactive member to the tissue.The ligand herein is any molecule or combination of molecules whichdemonstrates an affinity for a target. Examples of ligands includenucleic acid probes, antibodies, hapten conjugates, and cell adhesionpeptides such as RGD. The mechanisms involved in obtaining and usingsuch ligands are well-known. In embodiments, reactive members orcomplementary reactive members are incorporated into saccharides orpolysaccharides and metabolically incorporated into cells. See, e.g.,Baskin et al., supra.

Antibodies that specifically recognize antigens are useful in accordancewith one embodiment herein. Antibodies which are conjugated to areactive member are utilized to bind to proteins located on tissue.Monoclonal or polyclonal antibodies are raised against an antigen whichcan be any component of biological tissue and then purified usingconventional techniques. The term “antibody” is intended to includewhole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc.), andto include fragments thereof which are also specifically reactive with avertebrate, e.g., mammalian, protein. Antibodies may be fragmented usingconventional techniques and the fragments screened for utility in thesame manner as for whole antibodies. Thus, the term includes segments ofproteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of selectively reacting with acertain protein. Non-limiting examples of such proteolytic and/orrecombinant fragments include Fab, F(ab′)₂, Fab′, Fv, and single chainantibodies (scFv) containing a V[L] and/or V[H] domain joined by apeptide linker. The scFv's may be covalently or non-covalently linked toform antibodies having two or more binding sites. The present disclosureincludes polyclonal, monoclonal or other purified preparations ofantibodies and recombinant antibodies.

After purification, the ligands (e.g., antibodies, nucleic acid probes,hapten conjugates and cell adhesion peptides), are conjugated or linkedto reactive members or complementary reactive members in the mannersdescribed above. In addition, reactive members or complementary reactivemembers can be linked to ligands by cross-linking procedures which, inaccordance with the present invention, do not cause denaturing ormisfolding of the ligands. The terms “linked” or “conjugated” as usedherein are used interchangeably and are intended to include any or allof the mechanisms known in the art for coupling the reactive members orcomplementary reactive members to the ligand. For example, any chemicalor enzymatic linkage known to those with skill in the art iscontemplated including those which result from photoactivation and thelike. Homofunctional and heterobifunctional cross linkers are allsuitable. Reactive groups (distinguishable from reactive members orcomplementary reactive members herein) which can be cross-linked with across-linker include primary amines, sulfhydryls, carbonyls,carbohydrates and carboxylic acids.

Cross-linkers are conventionally available with varying lengths ofspacer arms or bridges. Cross-linkers suitable for reacting with primaryamines include homobifunctional cross-linkers such as imidoesters andN-hydroxysuccinimidyl (NHS) esters. Examples of imidoester cross-linkersinclude dimethyladipimidate, dimethylpimelimidate, anddimethylsuberimidate. Examples of NHS-ester cross-linkers includedisuccinimidyl glutamate, disucciniminidyl suberate andbis(sulfosuccinimidyl) suberate. Accessible amine groups present on theN-termini of peptides react with NHS-esters to form amides. NHS-estercross-linking reactions can be conducted in phosphate,bicarbonate/carbonate, HEPES and borate buffers. Other buffers can beused if they do not contain primary amines. The reaction of NHS-esterswith primary amines should be conducted at a pH of between about 7 andabout 9 and a temperature between about 4° C. and 30° C. for about 30minutes to about 2 hours. The concentration of NHS-ester cross-linkercan vary from about 0.1 to about 10 mM. NHS-esters are eitherhydrophilic or hydrophobic. Hydrophilic NHS-esters are reacted inaqueous solutions although DMSO may be included to achieve greatersolubility. Hydrophobic NHS-esters are dissolved in a water miscibleorganic solvent and then added to the aqueous reaction mixture.

Sulfhydryl reactive cross-linkers include maleimides, alkyl halides,aryl halides and a-haloacyls which react with sulfhydryls to form thiolether bonds and pyridyl disulfides which react with sulfhydryls toproduce mixed disulfides. Sulfhydryl groups on peptides and proteins canbe generated by techniques known to those with skill in the art, e.g.,by reduction of disulfide bonds or addition by reaction with primaryamines using 2-iminothiolane. Examples of maleimide cross-linkersinclude succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate andm-maleimidobenzoyl-N-hydroxysuccinimide ester. Examples of haloacetalcross-linkers include N-succinimidyl (4-iodoacetal) aminobenzoate andsulfosuccinimidyl (4-iodoacetal) aminobenzoate. Examples of pyridyldisulfide cross-linkers include1,4-Di-[3′-2′-pyridyldithio(propionamido)butane] andN-succinimidyl-3-(2-pyridyldithio)-propionate.

Carboxyl groups are cross-linked to primary amines or hydrazides byusing carbodimides which result in formation of amide or hydrazonebonds. In this manner, carboxy-termini of peptides or proteins can belinked. Examples of carbodiimide cross-linkers include1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride andN,N′-dicyclohexylcarbodiimide. Arylazide cross-linkers become reactivewhen exposed to ultraviolet radiation and form aryl nitrene. Examples ofarylazide cross-linkers include azidobenzoyl hydrazide and N-5-azido-2nitrobenzoyloxysuccinimide. Glyoxal cross linkers target the guanidylportion of arginine. An example of a glyoxal cross-linker isp-azidophenyl glyoxal monohydrate.

Heterobifunctional cross-linkers which possess two or more differentreactive groups are suitable for use herein. Examples includecross-linkers which are amine-reactive at one end andsulfhydryl-reactive at the other end such as4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene, N-succinimidyl3-(2-pyridyldithio)-propionate and the maleimide cross-linkers discussedabove.

Attachment of reactive members to the medical device functionalizes thedevice such that upon exposure to their complementary reactive memberswhich are situated on tissue, they are activated and form a covalentbond, thus adhering the device to the tissue. In one embodiment, alinker between the product of the reactive members or complementaryreactive members and the biological tissue is degradable by, e.g.,hydrolysis or enzymatic action. In this manner, the medical device canbe removable after a period of time. The degradable linkage may be,e.g., chelates or chemically or enzymatically hydrolyzable orabsorbable. Illustrative chemically hydrolyzable degradable linkagesinclude polymers, copolymers and oligomers of glycolide, dl-lactide,1-lactide, caprolactone, dioxanone, and tritnethylene carbonate.Illustrative enzymatically hydrolyzable biodegradable linkages includepeptidic linkages cleavable by metalloproteinases and collagenases.Additional illustrative degradable linkages include polymers andcopolymers of poly(hydroxy acid)s, poly(orthocarbonate)s,poly(anhydride)s, poly(lactone)s, poly(amino acid)s, poly(carbonate)s,poly(saccharide)s and poly(phosphonate)s. In certain embodiments, thedegradable linkage may contain ester linkages. Some non-limitingexamples include esters of succinic acid, glutaric acid, propionic acid,adipic acid, or amino acids, as well as carboxymethyl esters.

The medical device may be cut to a desired shape, packaged in single ordual pouches and sterilized by gamma or beta irradiation at 25-35 Kgy orby ethylene oxide. The ligand solution could be sterilized by theprevious cited method or by filtration in sterile conditions on 0.22 umfilter.

A kit for a medical device herein includes a polymeric medical devicesuch as a mesh or a patch which has a plurality of reactive members of aspecific binding pair attached to a surface of the device and acontainer which optionally functions as an applicator and is adapted tocontain a mixture including complementary reactive members of thespecific binding pair, the complementary reactive members having afunctionality that will adhere them to biological tissue upon contact.The kit may optionally include a container which contains a catalyst forcausing the reactive members of a specific binding pair to bind with thecomplementary reactive members of the specific binding pair. Thecatalyst may be a metal such as copper in solution. In embodiments, thekit contains a generator of microwaves or ultraviolet radiation.

It should be understood that variations can be made to the aboveembodiments that are with the purview of ordinary skill in the art. Forexample, other click chemistry reactions are suitable for use herein,e.g., Staudinger reaction of phosphines with alkyl azides. It iscontemplated that the above-described cross-linkers may be applied topolymers which make up the medical device to bind reactive members orcomplementary reactive members thereto. Accordingly, those skilled inthe art can envision modifications which are included within the scopeof the claimed invention that are not expressly set forth herein.

1. A method for bonding a polymeric medical device to biological tissuecomprising: providing a polymeric medical device having a plurality ofreactive members of a specific binding pair attached on a surface of themedical device; providing tissue having a plurality of complementaryreactive members of the specific binding pair; and contacting themedical device with the tissue, wherein upon contact of the reactivemembers on the surface of the medical device with the complimentaryreactive members on the tissue, covalent bonds are formed between thereactive members and the complementary reactive members, thus adheringthe medical device to the tissue.
 2. The method for bonding a polymericmedical device to tissue according to claim 1 wherein the members of thespecific binding pair bind to one another via a reaction selected fromthe group consisting of Huisgen cycloaddition reaction, a Diels-Alderreaction and a thiol-ene reaction.
 3. The method for bonding a polymericmedical device to tissue according to claim 2 wherein the members of thespecific binding pair are alkynes and azides.
 4. The method for bondinga polymeric medical device to tissue according to claim 3 wherein thereactive member is an alkyne and the complementary reactive member is anazide.
 5. The method for bonding a polymeric medical device to tissueaccording to claim 3 wherein the reactive members is an azide and thecomplementary reactive member is an alkyne.
 6. The method for bonding apolymeric medical device to tissue according to claim 2 wherein thereaction is catalyzed by copper to activate an alkyne and an azide for[3+2] cycloaddition.
 7. The method for bonding a polymeric medicaldevice to tissue according to claim 2 wherein the reaction involves acyclooctyne reagent and an azide for [3+2] cycloaddition.
 8. The methodfor bonding a polymeric medical device to tissue according to claim 2wherein the members of the specific binding pair are thiols and alkenes.9. The method for bonding a polymeric medical device to tissue accordingto claim 4 wherein the members of the specific binding pair are dienesand alkenes.
 10. The method for bonding a polymeric medical device totissue according to claim 2 wherein the tissue is provided withcomplementary reactive members of the specific binding pair by applyinga mixture or an aerosol containing the complementary reactive members tothe tissue, the complementary reactive members being conjugated to alinker adapted to link the complementary reactive members to the tissue.11. The method for bonding a polymeric medical device to tissueaccording to claim 10 wherein the complementary reactive members areattached to the tissue via an RGD linker.
 12. The method for bonding apolymeric medical device to tissue according to claim 10 wherein thecomplementary reactive members are attached to the tissue via aligand-receptor linkage.
 13. The method for bonding a polymeric medicaldevice to tissue according to claim 12 wherein the complementaryreactive members are conjugated to a linker selected from the groupconsisting of antibody, Fab, F(ab′)₂, Fv, single chain antibody (SCA)and single complementary-determining region (CDR).
 14. The method forbonding a polymeric medical device to tissue according to claim 10wherein the linker is degraded by hydrolysis or enzymatic action. 15.The method for bonding a polymeric medical device to tissue according toclaim 10 wherein the ligand binds to a receptor selected from the groupconsisting of peptides, oligosaccharides, oligonucleotides and lipids.16. The method for bonding a polymeric medical device to tissueaccording to claim 2 wherein the medical device is provided with thereactive members of the specific binding pair by surface modificationtechniques selected from the group consisting of plasma treatment,silane coupling treatment and acid sensitization.
 17. The method forbonding a polymeric medical device to tissue according to claim 1wherein the medical device is a mesh or a patch.
 18. The method forbonding a polymeric medical device to tissue according to claim 17wherein the patch is made of foam, woven material and non-wovenmaterial.
 19. The method for bonding a polymeric medical device totissue according to claim 1 wherein the medical device is made of apolymer selected from the group consisting of polycarbonates,polyolefins, polymethacrylates, polystyrenes, polyamides, polyurethanes,polyethylene terephthalate, poly (lactic acid), poly (glycolic acid),poly (hydroxbutyrate), dioxanones (e.g., 1,4-dioxanone),δ-valerolactone, 1,dioxepanones (e.g., 1,4-dioxepan-2-one and1,5-dioxepan-2-one), poly (phosphazine), polyesters, polyethyleneglycol, polyethylene oxides, polyacrylamides, cellulose esters,fluoropolymers, vinyl polymers, silk, collagen, alginate, chitin,chitosan, hyaluronic acid, chondroitin sufate,polyhydroxyethylmethylacrylate, polyvinylpyrrolidone, polyvinyl alcohol,polyacrylic acid, polyacetate, polycaprolactone, polypropylene,glycerols, poly(amino acids), copoly (ether-esters), polyalkyleneoxalates, polyamides, poly (iminocarbonates), polyalkylene oxalates,polyoxaesters, polyorthoesters, polyphosphazenes, polypeptides andcopolymers, block copolymers, homoploymers, blends and combinationsthereof.
 20. A kit comprising a polymeric medical device having aplurality of reactive members of a specific binding pair attached to asurface of the device and an container containing a mixture which can bea solution or suspension of complementary reactive members of thespecific binding pair, the complementary reactive members having afunctionality adapted to adhere them to biological tissue upon contact,and an applicator adapted to deliver the solution or suspension tobiological tissue.
 21. A kit according to claim 20 further comprising acontainer for containing a solution of a metal.
 22. A kit according toclaim 20 further comprising a generator for generating microwaves orultraviolet radiation.